When a binding assay (e.g., immunochromatography) that detects a test sample component using a porous solid phase (e.g., membrane) is performed, poor flow progression of the test sample in the porous solid phase may occur due to various reasons. For example, the poor flow progression of the test sample may occur when the test sample contains a substance (e.g., blood cell) that is larger than the pores of the porous solid phase and therefore cannot easily migrate/pass through the pores of the porous solid phase, or when a component that has been solubilized in the test sample becomes insoluble during the assay and clogs the porous solid phase. In such cases, the analytes, free labeled-antibodies, and the complexes of the analytes and the labeled antibodies, as well as other substances (e.g. oxidases) that are contained in the test sample and may have similar activities as the marker substances of the detection reagents, all of which are normally expected to migrate/pass freely through the porous solid phase, may cease to migrate/pass through it and may linger in undesired locations in the porous solid phase. As a result, the expected signal intensity normally associated with a presence of the analyte may be lost, which makes the detection difficult (making the assay impossible or causing false negative results), or non-specific reactions that interfere with the detection of the signals may occur (making the assay impossible or causing false positive results), so that the reliability or reproducibility of the test may deteriorate.
Conventionally, clogging of the porous solid phase has been prevented by removing impurities (e.g., a substance (e.g., blood cell) that is larger than the pores of the porous solid phase, or a component that easily becomes insoluble) contained in the test sample, by centrifugation or filtration or via precipitation, adsorption, or the like under specific conditions, before applying the test sample to the porous solid phase. However, each of the above manipulations is time-consuming, and a loss of the test sample is unavoidable during the manipulation. Moreover, these manipulations require extra costs. If the test sample contains infectious pathogenic microorganisms, the persons who carry out the test (e.g., physicians or researchers) may be subjected to a high risk of infection.
Methods that involve addition of surfactants to incubation media (e.g., Patent Document 1) have been widely used for the purpose of preventing non-specific interferences during the binding assays. However, depending on the type of the surfactant added, a capture reagent (e.g., antibody) bound to the solid-phase carrier may be removed from the carrier, or a binding reaction between the solid-phase carrier and the antibody may be hindered in the first place, so that the specific signal may decrease considerably and the desired detection sensitivity may not be achieved. Also, when the above method is applied to a binding assay that utilizes a porous solid phase, even if the surfactant (e.g., the glycoside surfactant disclosed in Patent Document 1) does not affect the detection sensitivity per se, the absolute amount of the test sample component in the assay system may be decreased due to dilution of the test sample, so that, in effect, the desired detection sensitivity may not be ensured. Moreover, Patent Document 1 only aims at preventing non-specific interferences. In Patent Document 1, poor flow progression of the test sample is neither mentioned nor suggested, and therefore it is not recognized as a problem to be solved.
In an immunoassay where whole blood is used as a test sample, the pretreatment of the test sample with centrifugation may be omitted if a blood cell separation pad is provided between the whole blood loading area and the porous solid phase (membrane) on the binding assay strip. According to this method, the blood cell components are retained in the separation pad and prevented from flowing into the porous solid phase over a short period of time, while the plasma components are allowed to flow into the immunochromatography membrane ahead of the blood cell components; thus the assay is not affected by the presence of the blood cell components.
Even in this case, however, poorness of sample flow progression (migration or passage) may still occur due to reasons other than clogging caused by the size of the impurities. Examples of these other reasons include a high viscosity of test sample, and drying of the porous solid phase during the assay. Highly viscous test samples (e.g., a test sample that contains a large amount of solid components such as blood cells or a large amount of proteins, or a test sample which has lost water due to evaporation) inherently have poor fluidity and therefore take long time to make progression in the porous solid phase, resulting in uneven progression and a lack of assay reproducibility.
Conventionally, when a test sample that contains solid components or has high viscosity (e.g., blood (whole blood), plasma, serum, saliva, sputum, or feces) is assayed by immunochromatography, it is necessary to dilute the test sample with an appropriate diluent (incubation medium) in advance. Phosphate buffered saline (PBS) or the like is used as the diluent, and bovine serum albumin (BSA) or the like is commonly added to the diluent in order to improve the dispersibility of the test sample. However, commercially available BSA may contain impurities such as immunoglobulin which may cause non-specific reactions. The problems associated with the inclusion of a surfactant in the diluent have been mentioned above. Also mentioned above is the fact that any attempts at solving the problems by modifying the constituents of the diluent will still suffer from the most fundamental problem, namely a reduction in the amount or concentration of the analyte available for the assay, caused by the dilution.
It has been reported (Patent Document 2) that, when a porous membrane for chromatographic assay is laminated for increasing its mechanical strength and for preventing evaporation (drying) of the fluid during the chromatographic assay, the porous membrane may become less hydrophilic due to the effect of an adhesive component used in the lamination, causing poor flow progression of the test sample. Patent Document 2 solves this poor flow progression problem by improving the hydrophilicity of the membrane based on an alternative choice of adhesive component along with a treatment of the membrane with an alkylsulfonic acid surfactant prior to lamination. However, Patent Document 2 does not recognize, or present a solution to, the problem of poor flow progression associated with test samples containing solid components or having high viscosity.
As a component in an enzyme immunoassay which uses a porous membrane as a solid-phase carrier, Patent Document 3 discloses a washing solution for the enzyme immunoassay solid phase that contains an alkyl glycoside surfactant or a steroid surfactant. The washing solution disclosed in Patent Document 3 is used for the purpose of washing off the test sample-derived substances that could cause non-specific reactions, or free marker substances, which have failed to pass/migrate through the porous membrane and thus remained on the membrane, after the test sample or the marker substances (which are capable of immunologically binding the analytes in the test sample) are added to the solid-phase carrier. The washing solution disclosed in Patent Document 3 is added to the solid-phase carrier separately from the test sample, and following the immunological reaction, for the purpose of removing the substances remaining on the porous membrane. Therefore, it requires a separate preparation step and a separate addition step independent of the test sample, making the assay procedure more complicated. The method of Patent Document 3 has been conceived by assuming a situation where some substances will remain on the membrane and need to be washed off, and thus it does not contain any consideration for preventing clogging of the membrane in the first place. Therefore, poor flow progression of the test sample, which is the problem addressed in the present invention, cannot be improved by the method disclosed in Patent Document 3.
A flow-through assay, such as the one described in the examples of Patent Document 3, may include a washing step. However, when testing a number of test samples at once, such an assay may require an auxiliary means such as pressurization and suction (e.g., increasing the volume of the water-absorbing pad under the membrane) in order to obtain reproducible results within a practical time frame, making the assay system less advantageous in terms of convenience and cost.
A method involving a use of a non-ionic surfactant for washing a solid substrate (array) on which physiologically active substances are immobilized has been disclosed (Patent Document 4). Patent Document 4 discloses an alkyl glucoside surfactant as the non-ionic surfactant. However, the invention of Patent Document 4 relates to an assay in which the array (having proteins or peptides immobilized thereon) is used for detecting the protein kinase activities contained in the lysates prepared from cultured cells, and its method involves washing the array prior to the detection in order to prevent the lysate components from being non-specifically adsorbed on the surface of the array substrate. Specifically, the method involves adding the surfactant-containing washing solution onto the array and then draining it from the array. Thus, the sole objective of Patent Document 4 is prevention of non-specific reactions, and similarly to Patent Document 1 discussed above, Patent Document 4 neither mentions nor suggests the issue of poor flow progression of test samples, not recognizing this issue as a problem to be solved.
In immunochromatography, the detection of the complex of the analyte and the detection reagent may be carried out by measuring the intensities of the reflected lights and calculating the absorbance (reflection absorbance) based on the measured intensities. The reflection absorbance is calculated from the ratio of the reflected light intensities measured at the measurement line and at its adjacent areas, but in this procedure, the measurement waveform (profile) may sometimes show disturbance near the measurement line (where the complex is detected) on the upstream or downstream side (particularly on the downstream side) due to an elevated intensity of the reflected light therein. When the concentration of the analyte contained in the test sample (specimen) is low, this disturbance of the measurement waveform cannot be ignored in determining the baseline. Thus, accuracy of detection of specific signals (peaks) may be lost, and the measurement itself may be rendered impossible. Since such disturbance of measurement waveform does not appear at a fixed frequency or to a fixed extent, the reproducibility of the assay is reduced, and an appropriate correction of the measured value (i.e. evaluation of the measurement waveform) may be necessary in each measurement.
In a common immunochromatography that uses a white membrane and optical detection means, the disturbance of measurement waveform may be recognized even with the naked eye, as a phenomenon in which the corresponding area on the membrane becomes whiter, as if bleached out, than the surrounding areas.
No prior arts have addressed the problem of the disturbance of the measurement waveform, and therefore no known solution exists for this problem.